Physiologically, the vertebrate immune system serves as a defense mechanism against invasion of the body by infectious objects such as microorganisms. Foreign proteins are effectively removed via the reticuloendothelial system by highly specific circulating antibodies, and viruses and bacteria are attacked by a complex battery of cellular and humoral mechanisms including antibodies, cytotoxic T lymphocytes (CTL), natural killer cells (NK), complement, etc. The leader of this battle is the T helper (TH) lymphocyte which, in collaboration with the Antigen Presenting Cells (APC), regulate the immune defense via a complex network of cytokines.
TH lymphocytes recognize protein antigens presented on the surface of the APC. They do not recognize, however, native antigen per se. Instead, they appear to recognize a complex ligand consisting of two components, a “processed” (fragmented) protein antigen (the so-called T cell epitope) and a Major Histocompatibility Complex class II molecule (O. Werdelin et al., 1 mm. Rev. 106, 181 (1988)). This recognition eventually enables the TH lymphocyte specifically to help B lymphocytes to produce specific antibodies towards the intact protein antigen (Werdelin et al., supra). A given T cell only recognizes a certain antigen-MHC combination and will not recognize the same or another antigen presented by a gene product of another MHC allele. This phenomenon is called MHC restriction.
Fragments of self-proteins are also presented by the APC, but normally such fragments are ignored or not recognized by the T helper lymphocytes. This is the main reason why individuals generally do not harbour autoantibodies in their serum eventually leading to an attack on the individual's own proteins (the so-called self- or autoproteins). However, in rare cases the process goes wrong, and the immune system turns towards the individual's own components, which may lead to an autoimmune disease.
The presence of some self-proteins is inexpedient in situations where they, in elevated levels, induce disease symptoms. Thus, tumour necrosis factor α (TNFα) is known to be able to cause cachexia in cancer patients and patients suffering from other chronic diseases (H. N. Langstein et al. Cancer Res. 51, 2302–2306, 1991). TNFα also plays important roles in the inflammatory process (W. P. Arend et al. Arthritis Rheum. 33, 305–315, 1990) and neutralization of TNFα by the use of monoclonal antibodies has thus been demonstrated to be beneficial in patients with chronic inflammatory diseases such as rheumatoid arthritis, Elliott et al., Lancet 1994, 344:1105–10 and Crohn's disease, van Dullemen et al., Gastroenterology 109(1):129–135(1995). There is therefore a need for a method for the induction of neutralizing antibodies against such TNFα proteins, and the present invention comprises a vaccine against TNFα which provide this property.
Tumour Necrosis Factor
1. General Background
Tumour necrosis factor (TNF) is a member of the cytokine family of regulatory proteins (see Walsh, G. and Headon, D. E., Protein Biotechnology, 1994, John Wiley & Sons Ltd.
England, p. 257–267). Two forms of TNF are now recognized, TNFα and TNFβ, respectively. Although both proteins bind the same receptors and elicit broadly similar biological responses, they are distinct molecules and share less than 30% homology. The original protein termed tumour necrosis factor, referred to as TNF, is more properly termed TNFα; it is also known as cachectin. TNFβ is also referred to as lymphotoxin.
TNFα is produced by a wide variety of cell types, most notably activated macrophages, monocytes, certain T lymphocytes and NK cells, in addition to brain and liver cells. The most potent known inducer of TNTα synthesis, is a complex biomolecule termed lipopolysaccharide. It contains both lipid and polysaccharide components, and is also referred to as endotoxin. Lipopolysaccharide itself is devoid of any anti-tumour activity. The serum of animals injected with lipopolysaccharide was found to contain a factor toxic to cancerous cells, and this factor, produced by specific cells in response to lipopolysaccharide, was termed tumour necrosis factor. Various other agents such as some viruses, fungi and parasites also stimulate the synthesis and release of this cytokine. Furthermore, TNFα may act in an autocrine manner, stimulating its own production.
Native human TNFα is a homotrimer, consisting of three identical polypeptide subunits tightly associated around a threefold axis of symmetry as will be further explained below. This arrangement resembles the assembly of protein subunits in many viral capsid proteins. The individual polypeptide subunits of human TNFα are non-glycosylated and consist of 157 amino acids. The molecule has a molecular weight of 17300 Da and contains a single intrachain disulphide linkage. Human TNFα is synthesized initially as a 233 amino acid precursor molecule. Proteolytic cleavage of the -76 to -1 presequence including a signal sequence releases native TNFα. TNFα may also exist in a 26000 Da membrane-bound form. Three TNFα monomeric subunits associate noncovalently to form a trimer as further explained below.
TNFα induces its biological effects by binding specific receptors present on the surface of susceptible cells. Two distinct TNFα receptors have been identified. One receptor (TNF-R55) has a molecular weight of 55000 Da, whereas the second receptor (TNF-R75) has a molecular weight of about 75000 Da. These two distinct receptor types show no more than 25% sequence homology. TNF-R55 is present on a wide range of cells, whereas the distribution of the TNF-R75 receptor is more limited. Both are transmembrane glycoproteins with an extracellular binding domain, a hydrophobic transmembrane domain and an intracellular effector domain.
The exact molecular mechanisms by which TNFα induces its biological effects remain to be determined. Binding of TNFα to its receptor seems to trigger a variety of events mediated by G-proteins in addition to the activation of adenylate cyclase, phospholipase A2 and protein kinases. The exact biological actions induced by TNFα may vary from cell type to cell type. Other factors, such as the presence of additional cytokines, further modulate the observed molecular effects attributed to TNFα action on sensitive cells.
The TNFα gene has been cloned and inserted in a variety of recombinant expression systems, both bacterial and eukaryotic. The resultant availability of large quantities of purified, biologically active TNFα has facilitated clinical evaluation a number of diseases, most notably cancer. Many such trials, using TNFα either alone or in combination with interferons, yielded, however, very disappointing results. Large quantities of TNFα can not be administered to patients owing to its toxic—if not lethal—side-effects.
As mentioned above prolonged production of inappropriately elevated levels of TNFα has also been implicated in the development of cachexia, the wasting syndrome often associated with chronic parasitic or other infections, and with cancer. TNFα is also involved in the metastasis and growth of certain tumours as well as in induction of anaemia. Furthermore, TNFα is also directly involved in the development of certain chronic inflammatory disorders in humans, including rheumatoid arthritis and Crohn's disease where administration of monoclonal anti-TNFα antibodies has been shown to be beneficial. TNFα is also involved in osteoporosis and Psoriasis. In addition, it has been shown in animal models that administration of anti-TNFα antibodies may decrease or prevent rejection of grafted or transplanted tissues Imagawa et al, Transplantation 51(1):57–62(1991).
2. Structure of TNFα
I. Introduction
The three-dimensional structure of human tumour necrosis factor (TNFα) has been solved (see “Tumor Necrosis Factors, Structure, Function and Mechanism of Action” edited by Bharat B. Aggarwal and Jan Vilcek, 1992 Marcel Dekker, Ind., New York, Chapter 5 “Crystal structure of TNFα”, by Jones, E. Y. Stuart, D. I. and Walker N. P. C.). The biological action of TNFα is dependent on its interaction with its receptors. These interactions are governed by the precise arrangement of the correctly folded tertiary structure. Thus, to understand how the TNFα molecule performs its biological function at the level of amino acid interactions, one must not only know the amino acid sequence, but also the three-dimensional structure.
II. Three-Dimensional Structure
Biologically active TNFα has been shown by analytical ultracentrifugation, small angle x-ray scattering, and gel electrophoresis to be in a trimer conformation in solution, and cross-linking studies have indicated that this is the active form of the protein (Smith and Baglioni, 1987). Analysis of circular dichroism spectra placed TNFα in the all-sheet class of proteins (Wingfield et al., 1987; Davis et al., 1987). Several different crystal forms have been reported for human recombinant TNFα. All the reported crystal forms exhibit crystallographic and/or non-crystallographic threefold symmetry indicative of the presence of TNFα as a trimer within the crystal.
The TNFα trimers lie in loosely packed arrays perforated by 100 Å diameter solvent channels. Only a small proportion of the molecular surface is involved in crystal packing contacts. Such contacts could slightly perturb a few side chains and perhaps even short portions of inherently flexible main chain from their preferred solution conformations.
A. Main-Chain Fold of the TNFα Monomer
The overall shape of a single 157-amino-acid subunit of the TNFα trimer is wedgelike with a height of approximately 55 Å and a maximum breadth of 35 Å near the base. The main-chain topology is illustrated in FIGS. 1a–c; it is essentially a β-sandwich structure formed by two antiparallel β-pleated sheets. The main-chainfold conforms to that of the classic jellyroll motif (FIG. 1c) (Richardson, 1981). The nomenclature adopted in FIG. 1 for the labels of the secondary structural units follows the established convention for viral structures. The standard eight β-strands (B to I) are all present but with an insertion between B and C that adds a short strand onto the edge of both β-sheets and truncates the N-terminal half of C, so that each β-pleated sheet contains five antiparallel β-strands, the back β-sheet comprising β-strands B′, B, I, D and G and the front sheet comprising β-strands C′, C, H, E and F.
The N terminus is highly flexible. This region, as far as residue 10 (see FIG. 1b), is rather independent of the rest of the molecule, with the first few residues free to sample a variety of conformations in the solvent. In contrast, the C terminus is embedded in the base of the back β-sheet and forms an integral part of this relatively flat secondary structural unit. The gradation in β-strand lengths and the insertion between β-strands B and C conspire to produce a front surface formed almost entirely of loops, and it is this “masked” side of the β-sandwich, which in the trimer is presented to the solvent. The crystallographic data yield a measure of the relative flexibility of the various parts of the structure. The β-strands form a fairly inflexible scaffold; in particular, the back β-sheet is situated at the core of the trimer and consequently is particularly rigid. As would be expected, it is the loops that adorn the outer solvent-accessible surface of the molecule, which exhibit high levels of flexibility/mobility. Overall, there is a general decrease in rigidity as the core becomes more loosely packed in the upper half of the molecule.
B. General Topology of the TNFα Trimer
Three TNFα monomeric subunits associate noncovalently to form a compact, conical trimer having a length of about 55 Å and a maximum breadth of 50 Å. The β-strands of the three individual β-sandwiches lie approximately parallel (the tilt is about 30°) to the threefold axis of the trimer. The interaction between subunits related by the three-fold axis is through a simple edge-to-face packing of the β-sandwich; the edge of the β-sandwich, consisting of strands F and G from one subunit, lies across the back β-sheet [GDIBB′] of a threefold related subunit (see FIG. 2). The carboxy termini lie close to the threefold axis.
The edge-to-face mode of packing produces an extremely tight association between the subunits. Thus the core of the trimer is completely inaccessible to solvent.
C. Amino Acid-Type Distribution
The overall distribution of residue types in the three-dimensional structure of TNFα echoes the general rule for proteins: namely, that hydrophobic residues cluster in the core of the molecule while charged residues decorate the surface. Thus the core of the TNFα sandwich has the expected filling of tightly intercalating large apolar residues.
The energetics of the system do not favour the existence of TNFα in a monomeric state. For a large interface area composed of complementary residues (e.g., polar residues matched against polar residues) the loss of solvent-accessible surface area confers a considerable energetic advantage to formation of the oligomer (i.e., the trimer). The exposure to solvent of the large patch of strongly hydrophobic residues normally buried in the lower portion of the trimeric interface would also act to destabilize the TNFα monomer.
3. Probes of Structure-Function
A. TNFα/Antibody Interactions
It has been observed that antibodies raised against TNFα from one species (e.g., human) do not cross-react with TNFα from another species (e.g., mouse) despite a sequence identity in excess of 80% and the ability of TNFα to bind to the TNFα receptors of other species. If the degree of sequence variation is mapped onto the three-dimensional structure, it is immediately apparent that the most sequence-variable regions of the molecule correspond to the antibody-accessible surface loops. The regions of highly conserved residues within the sandwich or at the trimeric interface are effectively invisible to antibodies. Thus the epitope for an antibody against TNFαwill always contain some residues that will vary between species, thus abolishing antibody binding. This implies that the characteristics of the interaction between TNFα and its receptor must somehow differ from those required for binding of an antibody to TNFα.
B. Site-Directed Mutagenesis
The role of various specific residues and regions of the TNFα molecule with regard to its biological (cytotoxic) activity and receptor binding has been probed by replacement of those residues by different amino acids or deletion of part of the sequence using the techniques of site-directed mutagenesis (Jones et al, p. 113–119).
The deletion of up to eight residues from the N-terminus without any deleterious effect on biological activity serves to emphasize the nonessential nature of this region for overall molecular stability. N-terminal residues appear to exert an indirect, second-order effect on the biological efficacy of the TNFα trimer.
Non-conservative substitutions of the normally highly conserved residues which form the tightly packed core of the β-sandwich distort the structure and hence abrogate the biological activity of TNFα (Yamagishi et al., 1989). Many such mutated proteins fail to form a stable, correctly folded molecule. Some conservative substitutions are permitted within the hydrophobic patch at the bottom of the threefold axis; however, there appears to be much greater leeway in the more loosely packed region near the top of the trimer. In particular, Cys 69 and Cys 101, which form the disulphide bridge between two connecting loops at the loosely packed top of the molecule, are relatively insensitive to changes (see FIG. 1a). Generally, however, in order to retain some biological activity of TNFα the mutations near the central axis of TNFαmust be highly conservative, preserving the overall shape of TNFα.
The residues on the surface of the molecule have a considerably greater freedom to mutate without incurring disastrous structural penalties as witnessed by the proliferation of variations of residues in this category between species. Thus drastic reductions in biological activity of TNFα due to substitutions in this area points to the direct involvement of such residues in the functional interaction of the TNFα trimer with its receptor. Residues comprising Arg 31, Arg 32 and Ala 33 situated in the connecting loop between the B and B′ strand of the back β sheet, Ser 86 and Tyr 87 situated in the connecting loop between the E and F strands of the front β-sheet, and Glu 146 situated in the connecting loop between the H strand of the front β sheet and the I-strand of the back β-sheet appear to be such amino acid residues (see FIG. 3). They appear to fall into two distinct regions on the front and the back sides of the TNFα monomer. The distribution of all deleterious mutations regardless of structural category further reinforces this picture. The existence of these “hot spots” for sensitivity of biological function to mutation has been reviewed by Yamagishi et al. (1989) and Goh et al. (1990).
4. Summary
A rich variety of data may now be brought to bear on the specific relationship of structure to function for TNFα. All available evidence points to the importance of the trimer as the stable natural unit. It is apparent that the two hotspot regions situated on separate sides of the TNFα monomer are brought close to each other in terms of neighbouring subunits in the trimer. Thus a region of functional importance consisting of residues 31 to 35, 84 to 87, and 143 to 148 appears to be located at the interface between two subunits on the lower half of the trimer. Yamagishi et al. (1989) report loss of receptor binding ability as well as cytotoxicity for the mutation of Asp 143 to Tyr, and Tsujimoto et al. (1987) report a similar effect for Arg 31 and Arg 32 to Asn and Thr. Thus the site may be associated directly with receptor binding as well as cytotoxicity. It is interesting that the receptor binding region of TNFα appears to lie at the interface between two subunits.
In summary, the detailed three-dimensional structure for TNFα serves to explain a wide range of observations on antibody binding, oligomerization, and site-directed mutagenesis. When the structure is considered in combination with recent, extensive site-directed mutants, a region of biological importance with regard to receptor binding is apparently at the subunits on the lower half of the trimer.